Understanding the mechanistic basis of retinal disease will require elucidation of the transcriptional regulatory networks (TRNs) which control gene expression during retinal development. Recent studies have shown that mutations in several transcription factors (TFs) controlling photoreceptor cell fate and differentiation underlie retinal disease in humans and mice. One such TF, Crx, not only causes cone-rod dystrophy when mutated, but a number of its downstream target genes have been implicated in retinal disease. In addition, a second TF required for normal rod differentiation, NRL, has been implicated in retinitis pigmentosa. The target genes of this latter TF largely remain to be identified. It is hypothesized that Crx and NRL lie near the top of a hierarchical TRN controlling rod photoreceptor differentiation. The overall goal of this proposal is to analyze the gene network controlled by the TFs, Crx and NRL, during mammalian rod photoreceptor development. These studies will serve to define the TRN topology of the rod cell, thereby laying the groundwork for future systems-level analyses of gene function in photoreceptor development and disease. Specific aim 1 will identify the target genes controlled by NRL as well as those coordinately regulated by Crx and NRL using microarray technology. Specific aim 2 will locate the putative cis-regulatory elements of these target genes employing bioinformatic approaches and test their function in vivo via electroporation of enhancer/reporter fusion constructs. Specific aim 3 will determine which of the putative target genes are direct targets of Crx and NRL by microarray analysis of retinae carrying transgenes overexpressing Crx and NRL in the presence or absence of protein-synthesis inhibitors. Specific aim 4 will determine the subsets of target genes controlled by TFs downstream of Crx and NRL using microarray analysis. Dr. Joseph Corbo, the Principal Investigator, is an M.D., Ph.D. with a doctoral degree in Biology and residency training in Anatomic Pathology and Neuropathology. He now seeks further training in genomics and cell biology under the mentorship of Dr. Constance L. Cepko, whose lab studies cell fate determination and differentiation in the retina. The candidate will combine these newly acquired skills with his clinical training in Neuropathology to pursue a career as a clinician-scientist applying cutting edge genomic technology to systems-level analyses of development and disease in the mammalian eye and brain.